Isothiazole and isoxazole compounds as transforming growth factor (TGF) inhibitors

ABSTRACT

Novel isothiazole and isoxazole compounds, including derivatives thereof, to intermediates for their preparation, to pharmaceutical compositions containing them and to their medicinal use are described. The compounds of the present invention are potent inhibitors of transforming growth factor (“TGF”)-β signaling pathway. They are useful in the treatment of various TGF-related disease states including, for example, cancer, and fibrotic diseases.

RELATED APPLICATIONS

This application claims benefit of priority under 35 U.S.C 119(e) toU.S. Provisional Application Nos. 60/412,131 filed on Sep. 18, 2002 and60/484,580 filed on Jul. 2, 2003, each of which is herein incorporatedin its entirety by reference.

BACKGROUND OF THE INVENTION

The present invention relates to novel isothiazole and isoxazolecompounds, including derivatives thereof, to intermediates for theirpreparation, to pharmaceutical compositions containing them and to theirmedicinal use. The compounds of the present invention are potentinhibitors of the transforming growth factor (“TGF”)-β signalingpathway. They are useful in the treatment of TGF-β related diseasestates including, for example, cancer and fibrotic diseases.

TGF-β activates both antiproliferative and tumor-promoting signalingcascades. Three mammalian TGF-β isoforms have been identified (TGF-βI,-βII, and -βIII). TGF-β production promotes tumor progression while itsblockade enhances antitumor activity. Blockade of TGF-β enhancesantitumor immune responses and inhibits metastasis. Thus there exists aneed in the art for compounds that inhibit the TGF-β signaling pathway.The present invention, as described below, answers such a need.

SUMMARY OF THE INVENTION

The present invention provides a novel compound containing a coreisothiazole or isoxazole ring substituted with at least one substitutedor unsubstituted 2-pyridyl moiety and at least one R¹ moiety as setforth herein, and all pharmaceutically acceptable salts, prodrugs,tautomers, hydrates and solvates thereof. In a compound of theinvention, the substituted or unsubstituted 2-pyridyl moiety and R¹moiety can be in an 1,2-, 1,3- or 1,4-relationship around the coreisothiazole or isoxazole ring; preferably, in an 1,2- or orthorelationship.

The present invention provides a compound of formula (A):

and all pharmaceutically acceptable salts, prodrugs, tautomers, hydratesand solvates thereof, where X, R¹, R³, R⁴, and s are each as set forthbelow.

In formula (A), as set forth above:

X is O or S;

R¹ is a saturated, unsaturated, or aromatic C₃–C₂₀ mono-, bi- orpolycyclic ring optionally containing at least one heteroatom selectedfrom the group consisting of N, O and S, wherein R¹ can optionally befurther independently substituted with at least one moiety independentlyselected from the group consisting of: carbonyl, halo, halo(C₁–C₆)alkyl,perhalo(C₁–C₆)alkyl, perhalo(C₁–C₆)alkoxy, (C₁–C₆)alkyl, (C₂–C₆)alkenyl,(C₂–C₆)alkynyl, hydroxy, oxo, mercapto, (C₁–C₆)alkylthio, (C₁–C₆)alkoxy,(C₅–C₁₀)aryl or (C₅–C₁₀)heteroaryl, (C₅–C₁₀)aryloxy or(C₅–C₁₀)heteroaryloxy, (C₅–C₁₀)ar(C₁–C₆)alkyl or(C₅–C₁₀)heteroar(C₁–C₆)alkyl, (C₅–C₁₀)ar(C₁–C₆)alkoxy or(C₅–C₁₀)heteroar(C₁–C₆)alkoxy, HO—(C═O)—, ester, amido ether, amino,amino(C₁–C₆)alkyl, (C₁–C₆)alkylamino(C₁–C₆)alkyl,di(C₁–C₆)alkylamino(C₁–C₆)alkyl, (C₅–C₁₀)heterocyclyl(C₁–C₆)alkyl,(C₁–C₆)alkyl- and di(C₁–C₆)alkylamino, cyano, nitro, carbamoyl,(C₁–C₆)alkylcarbonyl, (C₁–C₆)alkoxycarbonyl, (C₁–C₆)alkylaminocarbonyl,di(C₁–C₆)alkylaminocarbonyl, (C₅–C₁₀)arylcarbonyl,(C₅–C₁₀)aryloxycarbonyl, (C₁–C₆)alkylsulfonyl, and (C₅–C₁₀)arylsulfonyl;

preferably, R¹ can optionally be further independently substituted withzero to two moieties independently selected from the group consistingof, but not limited to, halo(C₁–C₆)alkyl, perhalo(C₁–C₆)alkyl,perhalo(C₁–C₆)alkoxy, (C₁–C₆)alkyl, (C₁–C₆)alkoxy,(C₅–C₁₀)ar(C₁–C₆)alkoxy or (C₅–C₁₀)heteroar(C₁–C₆)alkyloxy, amino,amino(C₁–C₆)alkyl, (C₁–C₆)alkylamino(C₁–C₆)alkyl,di(C₁–C₆)alkylamino(C₁–C₆)alkyl, and (C₅–C₁₀)heterocyclyl(C₁–C₆)alkyl;

each R³ is independently selected from the group consisting of:hydrogen, halo, halo(C₁–C₆)alkyl, (C₁–C₆)alkyl, (C₂–C₆)alkenyl,(C₂–C₆)alkynyl, perhalo(C₁–C₆)alkyl, phenyl, (C₅–C₁₀)heteroaryl,(C₅–C₁₀)heterocyclic, (C₃–C₁₀)cycloalkyl, hydroxy, (C₁–C₆)alkoxy,perhalo(C₁–C₆)alkoxy, phenoxy, (C₅–C₁₀)heteroaryl-O—,(C₅–C₁₀)heterocyclic-O—, (C₃–C₁₀)cycloalkyl-O—, (C₁–C₆)alkyl-S—,(C₁–C₆)alkyl-SO₂—, (C₁–C₆)alkyl-NH—SO₂—, O₂N—, NC—, amino,Ph(CH₂)₁₋₆HN—, (C₁–C₆)alkyl HN—, (C₁–C₆)alkylamino,[(C₁–C₆)alkyl]₂-amino, (C₁–C₆)alkyl-SO₂—NH—, amino(C═O)—, aminoO₂S—,(C₁–C₆)alkyl-(C═O)—NH—, (C₁–C₆)alkyl-(C═O)—[((C₁–C₆)alkyl)-N]—,phenyl-(C═O)—NH—, phenyl-(C═O)—[((C₁–C₆)alkyl)-N]—, (C₁–C₆)alkyl-(C═O)—,phenyl-(C═O)—, (C₅–C₁₀)heteroaryl-(C═O)—, (C₅–C₁₀)heterocyclic-(C═O)—,(C₃–C₁₀)cycloalkyl-(C═O)—, HO—(C═O)—, (C₁–C₆)alkyl-O—(C═O)—, H₂N(C═O)—,(C₁–C₆)alkyl-NH—(C═O)—, [(C₁–C₆)alkyl]₂-N—(C═O)—, phenyl-NH—(C═O)—,phenyl-[((C₁–C₆)alkyl)-N]—(C═O)—, (C₅–C₁₀)heteroaryl-NH—(C═O)—,(C₅–C₁₀)heterocyclic-NH—(C═O)—, (C₃–C₁₀)cycloalkyl-NH—(C═O)— and(C₁–C₆)alkyl-(C═O)—O—; preferably, R³ is hydrogen or (C₁–C₆)alkyl; morepreferably, R³ is hydrogen or methyl;

where alkyl, alkenyl, alkynyl, phenyl, heteroaryl, heterocyclic,cycloalkyl, alkoxy, phenoxy, amino of R³ is optionally substituted by atleast one substituent independently selected from (C₁–C₆)alkyl,(C₁–C₆)alkoxy, halo(C₁–C₆)alkyl, halo, H₂N—, Ph(CH₂)₁₋₆HN—, and(C₁–C₆)alkylHN—;

s is an integer from one to five; preferably, one to two; morepreferably, one; and

R⁴ is selected from the group consisting of: hydrogen, halo,halo(C₁–C₆)alkyl, (C₁–C₆)alkyl, (C₂–C₆)alkenyl, (C₂–C₆)alkynyl,perhalo(C₁–C₆)alkyl, phenyl, (C₅–C₁₀)heteroaryl, (C₅–C₁₀)heterocyclic,(C₃–C₁₀)cycloalkyl, hydroxy, (C₁–C₆)alkoxy, perhalo(C₁–C₆)alkoxy,phenoxy, (C₅–C₁₀)heteroaryl-O—, (C₅–C₁₀)heterocyclic-O—,(C₃–C₁₀)cycloalkyl-O—, (C₁–C₆)alkyl-S—, (C₁–C₆)alkyl-SO₂—,(C₁–C₆)alkyl-NH—SO₂—, O₂N—, NC—, amino, Ph(CH₂)₁₋₆NH—, alkylNH—,(C₁–C₆)alkylamino, [(C₁–C₆)alkyl]₂-amino, (C₁–C₆)alkyl-SO₂—NH—,amino(C═O)—, aminoSO₂—, (C₁–C₆)alkyl-(C═O)—NH—,(C₁–C₆)alkyl-(C═O)—((C₁–C₆)alkyl)-N]—, phenyl-(C═O)—NH—,phenyl-(C═O)—((C₁–C₆)alkyl)-N]—, (C₁–C₆)alkyl-(C═O)—, phenyl-(C═O)—,(C₅–C₁₀)heteroaryl-(C═O)—, (C₅–C₁₀)heterocyclic-(C═O)—,cycloalkyl-(C═O)—, HO—(C═O)—, (C₁–C₆)alkyl-O—(C═O)—, H₂N(C═O)—,(C₁–C₆)alkyl-NH—(C═O)—, ((C₁–C₆)alkyl)₂-N—(C═O)—, phenyl-NH—(C═O)—,phenyl-((C₁–C₆)alkyl)-N]—(C═O)—, (C₅–C₁₀)heteroaryl-NH—(C═O)—,(C₅–C₁₀)heterocyclic-NH—(C═O)—,

(C₃–C₁₀)cycloalkyl-NH—(C═O)— and (C₁–C₆)alkyl-(C═O)—O—; preferably, R⁴is hydrogen, (C₁–C₆)alkyl, or (C₃–C₁₀)cycloalkyl, where alkyl, alkenyl,alkynyl, phenyl, heteroaryl, heterocyclic, cycloalkyl, alkoxy, phenoxy,and amino of R⁴ is optionally substituted by at least one substituentindependently selected from the group consisting of (C₁–C₆)alkyl,(C₁–C₆)alkoxy, halo(C₁–C₆)alkyl, halo, H₂N—, Ph(CH₂)₁₋₆—NH—, and(C₁–C₆)alkylNH—.

In another embodiment of the invention, R¹ of formula (A), as set forthabove, is

where R^(2a) is as set forth herein.

In another embodiment of the invention, R¹ of formula (A), as set forthabove, is

In another embodiment of the invention, R¹ of formula (A), as set forthabove, is

In another embodiment of the invention, R¹ of formula (A), as set forthabove, is

In another embodiment of the invention, R¹ of formula (A), as set forthabove, is

where R^(2a) is as set forth herein.

In another embodiment of the invention, R¹ of formula (A), as set forthabove, is

where R^(2a) is as set forth herein.

In another embodiment of the invention, R¹ of formula (A), as set forthabove, is

where R^(2a) is as set forth herein.

Each of R¹ above can optionally be further substituted by at least oneR^(2a) group, as set forth herein.

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

where R^(2a) is as set forth herein.

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

where R^(2a) is as set forth herein.

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

where R^(2a) is as set forth herein.

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

where R^(2a) is as set forth herein.

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

where R^(2a) is as set forth herein.

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

where R^(2a) is as set forth herein.

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

where R^(2a) is as set forth herein.

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

where R^(2a) is as set forth herein.

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention, R¹ of formula (Ia), as set forthabove, is

In another embodiment of the invention, R¹ of formula (A), as set forthabove, is

where R^(2a) is as set forth herein and where the proviso language doesnot apply.

In another embodiment of the invention, R¹ of formula (A), as set forthabove, is selected from the group consisting of:

and where the proviso language does not apply.

In another embodiment of the invention R¹ of formula (A), as set forthabove, is selected from the group consisting of:

and where the proviso language does not apply.

In another embodiment of the invention, R¹ of formula (A), as set forthabove, is selected from the group consisting of:

and

where R^(2a) is as set forth herein and where the proviso language doesnot apply.

The invention also provides a pharmaceutical composition comprising atleast one compound of the invention and a pharmaceutically acceptablecarrier.

The invention further provides a method of preparation of a compound ofthe invention.

The invention still further provides a method of preventing or treatinga TGF-related disease state in an animal or human comprising the step ofadministering a therapeutically effective amount of at least onecompound of the invention to the animal or human suffering from theTGF-related disease state.

The invention still further provides the use of a compound of theinvention in the preparation of a medicament for the prevention ortreatment of a TGF-related disease state in an animal or human.

Definitions

As used herein, the article “a” or “an” refers to both the singular andplural form of the object to which it refers.

As used herein, the term “alkyl,” as well as the alkyl moieties of othergroups referred to herein (e.g., alkoxy) refers to a linear or branchedsaturated hydrocarbon (e.g., methyl, ethyl, n-propyl, isopropyl,n-butyl, iso-butyl, secondary-butyl, tertiary-butyl).

As used herein, the term “cycloalkyl” refers to a mono or bicycliccarbocyclic ring (e.g., cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclopentenyl,cyclohexenyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl andbicyclo[5.2.0]nonanyl).

As used herein, the term “halogen” or “halo” refers to fluoro, chloro,bromo or iodo or fluoride, chloride, bromide or iodide.

As used herein, the term “halo-substituted alkyl” or “haloalkyl” refersto an alkyl radical, as set forth above, substituted with one or morehalogens, as set forth above, including, but not limited to,chloromethyl, dichloromethyl, fluoromethyl, difluoromethyl,trifluoromethyl, and 2,2,2-trichloroethyl.

As used herein, the term “perhaloalkyl” refers to an alkyl radical, asset forth above, where each hyrdrogen of the alkyl group is replacedwith a “halogen” or “halo”, a set forth above.

As used herein, the term “alkenyl” refers to a linear or branchedhydrocarbon chain radical containing at least two carbon atoms and atleast one double bond. Examples include, but are not limited to,ethenyl, 1-propenyl, 2-propenyl (allyl), iso-propenyl,2-methyl-1-propenyl, 1-butenyl, and 2-butenyl.

As used herein, the term “alkynyl” refers to a linear or branchedhydrocarbon chain radical having at least one triple bond including, butnot limited to, ethynyl, propynyl, and butynyl.

As used herein, the term “carbonyl” refers to a >C═O moiety.Alkoxycarbonylamino (i.e. alkoxy(C═O)—NH—) refers to an alkyl carbamategroup. The carbonyl group is also equivalently defined herein as (C═O).

As used herein, the term “phenyl-[(alkyl)-N]—(C═O)—” refers to aN.N′-disubstituted amide group of the formula

As used herein, the term “aryl” refers to an aromatic radical such as,for example, phenyl, naphthyl, tetrahydronaphthyl, and indanyl.

As used herein, the term “heteroaryl” refers to an aromatic groupcontaining at least one heteroatom selected from O, S and N. Forexample, heteroaryl groups include, but are not limited to, pyridyl,pyrazinyl, pyrimidinyl, pyridazinyl, thienyl, furyl, imidazolyl,pyrrolyl, oxazolyl (e.g., 1,3-oxazolyl, 1,2-oxazolyl), thiazolyl (e.g.,1,2-thiazolyl, 1,3-thiazolyl), pyrazolyl, tetrazolyl, triazolyl (e.g.,1,2,3-triazolyl, 1,2,4-triazolyl), oxadiazolyl (e.g.,1,2,3-oxadiazolyl), thiadiazolyl (e.g., 1,3,4-thiadiazolyl), quinolyl,isoquinolyl, benzothienyl, benzofuryl, and indolyl.

As used herein, the term “heterocyclic” refers to a saturated orunsaturated C₃–C₂₀ mono-, bi- or polycyclic group containing at leastone heteroatom selected from N, O, and S. Examples of heterocyclicgroups include, but are not limited to, azetidinyl, tetrahydrofuranyl,imidazolidinyl, pyrrolidinyl, piperidinyl, piperazinyl, oxazolidinyl,thiazolidinyl, pyrazolidinyl, thiomorpholinyl, tetrahydrothiazinyl,tetrahydro-thiadiazinyl, morpholinyl, oxetanyl, tetrahydrodiazinyl,oxazinyl, oxcithiazinyl, indolinyl, isoindolinyl, quincuclidinyl,chromanyl, isochromanyl, benzocazinyl, and the like. Examples ofmonocyclic saturated or unsaturated ring systems aretetrahydrofuran-2-yl, tetrahydrofuran-3-yl, imidazolidin-1-yl,imidazolidin-2-yl, imidazolidin-4-yl, pyrrolidin-1-yl, pyrrolidin-2-yl,pyrrolidin-3-yl, piperidin-1-yl, piperidin-2-yl, piperidin-3-yl,piperazin-1-yl, piperazin-2-yl, piperazin-3-yl, 1,3-oxazolidin-3-yl,isothiazolidine, 1,3-thiazolidin-3-yl, 1,2-pyrazolidin-2-yl,1,3-pyrazolidin-1-yl, thiomorpholin-yl, 1,2-tetrahydrothiazin-2-yl,1,3-tetrahydrothiazin-3-yl, tetrahydrothiadiazin-yl, morpholin-yl,1,2-tetrahydrodiazin-2-yl, 1,3-tetrahydrodiazin-1-yl, 1,4-oxazin-2-yl,and 1,2,5-oxathiazin-4-yl.

As used herein, the term “pharmaceutically acceptable acid additionsalt” refers to non-toxic acid addition salts, i.e., salts derived frompharmacologically acceptable anions, such as the hydrochloride,hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acidphosphate, acetate, lactate, citrate, acid citrate, tartrate,bitartrate, succinate, maleate, fumarate, gluconate, saccharate,benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate,p-toluenesulfonate and pamoate [i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)]salts.

As used herein, the term “pharmaceutically acceptable base additionsalt” refers to non-toxic base addition salts, i.e., salts derived fromsuch pharmacologically acceptable cations such as alkali metal cations(e.g. potassium and sodium) and alkaline earth metal cations (e.g.,calcium and magnesium), ammonium or water-soluble amine addition saltssuch as N-methylglucamine-(meglumine), and the lower alkanolammonium andother base salts of pharmaceutically acceptable organic amines.

As used herein, the term “suitable substituent”, “substituent” or“substituted” refers to a chemically and pharmaceutically acceptablefunctional group, i.e., a moiety that does not negate the inhibitoryand/or therapeutic activity of the inventive compounds. Such suitablesubstituents may be routinely selected by those skilled in the art.Illustrative examples of suitable substituents include, but are notlimited to, carbonyl, halo, haloalkyl, perfluoroalkyl, perfluoroalkoxy,alkyl, alkenyl, alkynyl, hydroxy, oxo, mercapto, alkylthio, alkoxy, arylor heteroaryl, aryloxy or heteroaryloxy, aralkyl or heteroaralkyl,aralkoxy or heteroaralkoxy, HO—(C═O)—, ester, amido, ether, amino,alkyl- and dialkylamino, cyano, nitro, carbamoyl, alkylcarbonyl,alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylcarbonyl,aryloxycarbonyl, alkylsulfonyl, arylsulfonyl and the like. Those skilledin the art will appreciate that many substituents can be substituted byadditional substituents.

As used herein, the term “TGF-related disease state” refers to anydisease state mediated by the production of TGF-β.

As used herein, the term “Ph” refers to phenyl.

As used herein, the term “a saturated, unsaturated, or aromatic C₃–C₂₀mono-, bi- or polycyclic ring optionally containing at least oneheteroatom” refers to, but is not limited to,

where R^(2a) is independently selected from the group consisting of:(C₁–C₆)alkyl, (C₂–C₆)alkenyl, (C₂–C₆)alkynyl, (C₃–C₁₀)cycloalkyl,(C₅–C₁₀)aryl, (C₁–C₆)alkylaryl, amino, carbonyl, carboxyl, (C₂–C₆)acid,(C₁–C₆)ester, (C₅–C₁₀)heteroaryl, (C₅–C₁₀)heterocyclyl, (C₁–C₆)alkoxy,nitro, halo, hydroxyl, (C₁–C₆)alkoxy(C₁–C₆)ester, and those groupsdescribed in U.S. application Ser. Nos. 10/094,717, 10/094,760, and10/115,952, each of which is herein incorporated in its entirety byreference; and where alkyl, alkenyl, alkynyl, cycloalkyl, aryl, amino,acid, ester, heteroaryl, heterocyclyl, and alkoxy of R^(2a) isoptionally substituted by at least one moiety independently selectedfrom the group consisting of halo, (C₁–C₆)alkyl, (C₂–C₆)alkenyl,(C₂–C₆)alkynyl, perhalo(C₁–C₆)alkyl, phenyl, (C₃–C₁₀)cycloalkyl,(C₅–C₁₀)heteroaryl, (C₅–C₁₀)heterocyclic, formyl, NC—,(C₁–C₆)alkyl-(C═O)—, phenyl-(C═O)—, HO—(C═O)—, (C₁–C₆)alkyl-O—(C═O)—,(C₁–C₆)alkyl-NH—(C═O)—, ((C₁–C₆)alkyl)₂-N—(C═O)—, phenyl-NH—(C═O)—,phenyl-[((C₁–C₆)alkyl)-N]—(C═O)—, O₂N—, amino, (C₁–C₆)alkylamino,((C₁–C₆)alkyl)₂-amino, (C₁–C₆)alkyl-(C═O)—NH—,(C₁–C₆)alkyl-(C═O)—[((C₁–C₆)alkyl)-N]—, phenyl-(C═O)—NH—,phenyl-(C═O)—[((C₁–C₆)alkyl)-N]—, H₂N—(C═O)—NH—,(C₁–C₆)alkyl-HN—(C═O)—NH—, ((C₁–C₆)alkyl)₂N—(C═O)—NH—,(C₁–C₆)alkyl-HN—(C═O)—[((C₁–C₆)alkyl)-N]—,((C₁–C₆)alkyl)₂N—(C═O)—[(C₁–C₆)alkyl-N]—, phenyl-HN—(C═O)—NH—,(phenyl)₂N—(C═O)—NH—, phenyl-HN—(C═O)—[((C₁–C₆)alkyl)-N]—,(phenyl-)₂N—(C═O)—[((C₁–C₆)alkyl)-N]—, (C₁–C₆)alkyl-O—(C═O)—NH—,(C₁–C₆)alkyl-O—(C═O)—[((C₁–C₆)alkyl)-N]—, phenyl-O—(C═O)—NH—,phenyl-O—(C═O)-[(alkyl)-N]—, (C₁–C₆)alkyl-SO₂NH—, phenyl-SO₂NH—,(C₁–C₆)alkyl-SO₂—, phenyl-SO₂—, hydroxy, (C₁–C₆)alkoxy,perhalo(C₁–C₆)alkoxy, phenoxy, (C₁–C₆)alkyl-(C═O)—O—,(C₁–C₆)ester-(C₁–C₆)alkyl-O—, phenyl-(C═O)—O—, H₂N—(C═O)—O—,(C₁–C₆)alkyl-HN—(C═O)—O—, ((C₁–C₆)alkyl)₂N—(C═O)—O—, phenyl-HN—(C═O)—O—,and (phenyl)₂N—(C═O)—O—.

DETAILED DESCRIPTION OF THE INVENTION

The following reaction schemes illustrate the preparation of thecompounds of the present invention. A compound of the invention may beprepared by methods analogous to those described in U.S. applicationSer. Nos. 10/094,717, 10/094,760, and 10/115,952 and WO 02/40476. Unlessotherwise indicated, X, R¹, R³, R⁴, R^(2a), and s in the reactionschemes and the discussion that follow are defined above.

Scheme 1 refers to the preparation of compounds of the formula (A) whereX is O. Referring to Scheme 1, a compound of the formula III wasprepared from aldehydes of the formula II by first treatment with anaromatic amine, such as aniline, in a polar solvent. Suitable solventsinclude ethyl acetate, isopropyl acetate, or tetrahydrofuran, preferablyisopropyl acetate. The resulting reaction mixture was heated to atemperature from about 50° C. to about 100° C., preferably 60° C., andthen slowly treated with phosphorous acid diphenyl ester. Thetemperature of the reaction mixture was maintained for a period fromabout 30 minutes to about 3 hours, preferably 1 hour and then cooled toambient temperature overnight. A compound of formula II was preparedaccording to Preparation E, set forth below.

A compound of the formula V was prepared from a compound of the formulaIII by reaction with an aldehyde of the formula IV in the presence of abase, such as potassium tert-butoxide, in a polar solvent. Suitablesolvents include ethyl acetate, isopropyl acetate, or tetrahydrofuran,preferably a mixture of tetrahydrofuran and isopropyl acetate. Theaforesaid reaction was run at a temperature from about 0° C. to about100° C., preferably about 22° C. (ambient temperature), for a periodfrom about 30 minutes to about 5 hours, preferably 2 hours. Theresulting reaction mixture was then treated with acid, such ashydrochloric acid for a period from about 30 minutes to about 5 hours,preferably about 1 hour. A compound of formula (A) where X=O wasprepared from a compound of formula V by treatment with an excess ofhydroxyl amine in a polar solvent such as ethanol, at a temperature ofabout 0° C. to about 60° C., preferably about room temperature. Theresulting adduct was then stirred in N,N-dimethylformamidedimethylacetal and heated neat at a temperature from about 60° C. toabout 100° C., preferably about 80° C., for a period of about 60 minutesto about 60 hours, preferably about 36 hours.

Scheme 2 refers to the preparation of compounds of the formula V, whichare intermediates in the preparation of compounds of formula (A) inScheme 1. Referring to Scheme 2, a compound of the formula V wasprepared from a compound of the formula VI by treatment with a base,such as butyl lithium, at a temperature of about −60° C. for a timeperiod of about 90 minutes, followed by the slow addition of pyridylamide of the formula VII, which is either commercially available orprepared according to methods analogous to those of Preparation C, asset forth below, where R¹ is replaced by

in a polar aprotic solvent, such as tetrahydrofuran. The aforesaidreaction was run at a temperature from about −78° C. to about 0° C.,preferably about −20° C., for a period from about 1 hour to about 10hours, preferably about 3 hours.

Alternatively the compound of formula V is prepared according to themethods of Davies, I. W.; Marcoux, J. F.; Corley, E. G.; Journet, M.;Cai, D.-W.; Palucki, M.; Wu, J.; Larsen, R. D.; Rossen, K.; Pye, P. J.;DiMichele, L.; Dormer, P.; Reider, P. J.; J. Org. Chem., Vol. 65, pp.8415–8420 (2000).

Scheme 3 refers to the preparation of compounds of the formula (A) whereR¹ is

Referring to Scheme 3, compounds of the formula IX were prepared fromcompounds of the formula VIII, both of which are either commerciallyavailable or can be prepared according to the procedure described inPreparation D, each as set forth below. In Scheme 3 the compounds offormula (A) was prepared from compound IX according to proceduresdescribed in Scheme 1.

Scheme 4 refers to the preparation of compounds of the formula V, whichare intermediates in the preparation of compounds of formula (A) inScheme 1. Referring to Scheme 4, a compound of the formula V wasprepared from compound of the formula X, which is either commerciallyavailable or prepared according to Preparation B, as set forth below, byreaction with a compound of the formula R¹—Cl, in the presence of acatalyst such as palladium II acetate, a base (e.g., potassiumtert-butoxide, and AMPHOS® (i.e.,2-dicyclohexylphosphino-2′-(N,N-dimethylamino)biphenyl, commerciallyavailable from Strem Chemicals, Newburyport, Mass.)) in a polar aproticsolvent such as tetrahydrofuran. The aforesaid reaction was run at atemperature from about 50° C. to about 100° C., preferably about 75° C.,for a period from about 6 hours to about 24 hours, preferably about 18hours.

Scheme 5 refers to the preparation of compounds of the formula A, whereX=S. Referring to Scheme 5, an amide of the formula XXIX can be preparedfrom compounds of the formula XV (commercially available from AldrichChemical, Milwaukee, Wis.) by treatment with thionyl chloride followedby quenching with ammonia. Thioamide XXX can be prepared from XXIX bytreatment with Lawson's reagent in a solvent such as toluene, attemperatures of 20–100° C. A compound of formula (A) where X=S could beprepared from XXX as described by Tetrahedron Letters, 25, 409–410,1984, by reaction with acetylene XXXI. A compound of formula XXXI can beprepared from R¹br by combination with a catalyst such as PDCl2(PPh)2,in the presence of an additive such as CuI, andethynyl-trimethyl-silane, at a temperature of about 0° C. to about 80°C., preferably about room temperature, in a polar solvent such astetrahydrofuran. The trimethyl-silane group can then be removed understandard deprotection conditions.

Preparation A refers to the preparation of compounds of the formula IV,which are intermediates useful in the preparation of compounds of theformula (A). In Preparation A, R is a simple alkyl group such as methylor ethyl. Referring to Preparation A, compounds of the formula XII wereprepared from a compound of the formula XI, wherein X is a chloride orbromide, by an alkoxycarbonylation reaction. Suitable conditions includemetal-halogen exchange with butyl lithium in a solvent such astetrahydrofuran at a temperature of about 0° C., for a period of time ofabout 30 minutes, followed by the addition of ethylchloroformate at atemperature of about 0° C., followed by a period of time of about 2.4hours at about 50° C. A compound of formula XI are commerciallyavailable.

The compound of the formula IV was prepared from a compound of theformula XII by a two-step process. First the compound of formula XII wastreated with a reducing agent. Suitable reducing agents include lithiumborohydride, sodium borohydride, lithium aluminum hydride, and borane intetrahydrofuran. Suitable solvents for the aforesaid reaction includemethanol, ethanol, tetrahydrofuran, diethyl ether, and dioxane. Theaforesaid reaction was run at a temperature from about 0° C. to about100° C., preferably about 65° C., for a period from about 10 minutes toabout 1 hour, preferably about 30 minutes. The resulting primary alcoholwas then oxidized to the corresponding aldehyde of the formula IV bytreatment with an oxidizing agent, such as N-methyl morpholineN-oxide/TPAP, Dess-Martin reagent, PCC or oxalyl chloride-DMSO,preferably oxalyl chloride-DMSO. Suitable solvents for the aforesaidreaction include chloroform, tetrahydrofuran, or dichloromethane. Theaforesaid reaction was conducted at a temperature from about −78° C. toabout 22° C. for a time from about 15 minutes to about 3 hours,preferably about 1 hour.

Preparation B refers to the preparation of compounds of the formula X,which are intermediates useful in the preparation of compounds of theformula (A). Referring to Preparation B, a compound of formula XIII wasprepared from a compound of the formula II by reaction with methylmagnesium bromide in a polar solvent such as a mixture oftetrahydrofuran and toluene. The aforesaid reaction was run at atemperature from about −78° C. to about 0° C., preferably about −60° C.,for a period from about 10 minutes to about 1 hour, preferably about 40minutes, followed by a period of about 90 minutes at a temperature ofabout −10° C. A compound of formula II was prepared according toPreparation E, set forth below.

The compound of formula X was prepared from a compound of the formulaXIII by treatment with an oxidizing agent, such as N-methyl morpholineN-oxide/TPAP, Dess-Martin reagent, PCC or oxalyl chloride-DMSO,preferably oxalyl chloride-DMSO. Suitable solvents for the aforesaidreaction include chloroform, tetrahydrofuran, or dichloromethane. Theaforesaid reaction was conducted at a temperature from about −78° C. toabout 22° C. for a time from about 15 minutes to about 3 hours,preferably about 1 hour.

Preparation C refers to the preparation of compounds of the formula VII,which are intermediates useful in the preparation of compounds of theformula (A). In Preparation C, R is a simple alkyl group such as methylor ethyl. Referring to Preparation C, compounds of the formula XV wereprepared from a compound of the formula XIV, which may be preparedaccording to a procedure described in Preparation A or are commerciallyavailable, by treatment with a base such as lithium hydroxide, in apolar protic solvent. Suitable solvents for the aforesaid reactionincluded methanol, ethanol, and water. The aforesaid reaction wasconducted at a temperature from about 0° C. to about 30° C., preferablyabout 22° C. (room temperature) for a time from about 15 minutes toabout 3 hours, preferably about 1 hour.

The compound of the formula VII was prepared from a compound of theformula XV by reaction with a suitable activating agent and a compoundof the formula

and a base. Suitable activating agents included thionyl chloride,carbonyldiimidazole, EDCI and DCC, preferably oxalyl chloride. Suitablebases included triethylamine, Hunig's base, or DBU, preferablytriethylamine. Suitable solvents for the aforesaid reaction includemethylene chloride, N,N′-dimethylformamide, tetrahydrofuran, and amixture thereof, preferably methylene chloride. The aforesaid reactionwas conducted at a temperature from about 0° C. to about 30° C.,preferably about 22° C. (room temperature) for a time from about 6 hoursto about 48 hours, preferably about 12 hours.

Preparation D refers to the preparation of compounds of the formulaVIII, which is an intermediate useful in the preparation of compounds offormula (A), where R¹ is

In Preparation D, R is (C₁–C₆)alkyl. The compound of formula VIII wasprepared from a compound of formula XVI by treatment with an alkylhalide, such as methyl iodide, in the presence of a base such as sodiumhydride, in a polar aprotic solvent such as N,N′-dimethylformamide.Compounds of the formula XVI are commercially available.

Preparation E refers to the preparation of compounds of the formula II,which are intermediates useful in the preparation of compounds offormula (A), as set forth above. In Preparation E, R is a simple alkylgroup such as methyl or ethyl. Referring to Preparation E, compounds ofthe formula XVIII were prepared from heteroarylhalides of the formulaXVII, wherein X is a chloride or bromide, according to the proceduredescribed for the preparation of compound XII from compound XI inPreparation A. The compound of the formula II was prepared from acompound of the formula XVIII according to the two-step processdescribed for the preparation of compound IV from compound XII inPreparation A.

All pharmaceutically acceptable salts, prodrugs, tautomers, hydrates andsolvates of a compound of the invention is also encompassed by theinvention.

A compound of the invention which is basic in nature is capable offorming a wide variety of different salts with various inorganic andorganic acids. Although such salts must be pharmaceutically acceptablefor administration to animals and humans, it is often desirable inpractice to initially isolate a compound of the invention from thereaction mixture as a pharmaceutically unacceptable salt and then simplyconvert the latter back to the free base compound by treatment with analkaline reagent, and subsequently convert the free base to apharmaceutically acceptable acid addition salt. The acid addition saltsof the base compounds of this invention are readily prepared by treatingthe base compound with a substantially equivalent amount of the chosenmineral or organic acid in an aqueous solvent medium or in a suitableorganic solvent such as, for example, methanol or ethanol. Upon carefulevaporation of the solvent, the desired solid salt is obtained.

The acids which can be used to prepare the pharmaceutically acceptableacid addition salts of the base compounds of this invention are thosewhich form non-toxic acid addition salts, i.e., salts containingpharmacologically acceptable anions, such as chloride, bromide, iodide,nitrate, sulfate or bisulfate, phosphate or acid phosphate, acetate,lactate, citrate or acid citrate, tartrate or bitartrate, succinate,maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate andpamoate [i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)] salts.

A compound of the invention which is also acidic in nature, e.g.,contains a COOH or tetrazole moiety, is capable of forming base saltswith various pharmacologically acceptable cations. Although such saltsmust be pharmaceutically acceptable for administration to animals andhumans, it is often desirable in practice to initially isolate acompound of the invention from the reaction mixture as apharmaceutically unacceptable salt and then simply convert the latterback to the free acid compound by treatment with an acidic reagent, andsubsequently convert the free acid to a pharmaceutically acceptable baseaddition salt. Examples of such pharmaceutically acceptable baseaddition salts include the alkali metal or alkaline-earth metal saltsand particularly, the sodium and potassium salts. These salts can beprepared by conventional techniques. The chemical bases which can beused as reagents to prepare the pharmaceutically acceptable baseaddition salts of this invention are those which form non-toxic basesalts with the herein described acidic compounds of the invention. Thesenon-toxic base salts include salts derived from such pharmacologicallyacceptable cations as sodium, potassium, calcium and magnesium, etc.These salts can easily be prepared by treating the corresponding acidiccompounds with an aqueous solution containing the desiredpharmacologically acceptable cations, and then evaporating the resultingsolution to dryness, preferably under reduced pressure. Alternatively,they may also be prepared by mixing lower alkanolic solutions of theacidic compounds and the desired alkali metal alkoxide together, andthen evaporating the resulting solution to dryness in the same manner asbefore. In either case, stoichiometric quantities of reagents arepreferably employed in order to ensure completeness of reaction andmaximum product yields.

Isotopically-labeled compounds are also encompassed by the presentinvention. As used herein, an “isotopically-labeled compound” refers toa compound of the invention including pharmaceutical salts, prodrugsthereof, each as described herein, in which one or more atoms arereplaced by an atom having an atomic mass or mass number different fromthe atomic mass or mass number usually found in nature. Examples ofisotopes that can be incorporated into compounds of the inventioninclude isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous,fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P,³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively.

By isotopically-labeling a compound of the present invention, thecompounds may be useful in drug and/or substrate tissue distributionassays. Tritiated (³H) and carbon-14 (¹⁴C) labeled compounds areparticularly preferred for their ease of preparation and detectability.Further, substitution with heavier isotopes such as deuterium (² H) canafford certain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements and, hence, may be preferred in some circumstances.Isotopically labeled compounds of the invention, includingpharmaceutical salts, prodrugs thereof, can be prepared by any meansknown in the art.

Stereoisomers (e.g., cis and trans isomers) and all optical isomers of acompound of the invention (e.g., R and S enantiomers), as well asracemic, diastereomeric and other mixtures of such isomers arecontemplated by the present invention.

The compounds, salts, prodrugs, tautomers, hydrates, and solvates of thepresent invention can exist in several tautomeric forms, including theenol and imine form, and the keto and enamine form and geometric isomersand mixtures thereof. All such tautomeric forms are included within thescope of the present invention. Tautomers exist as mixtures of atautomeric set in solution. In solid form, usually one tautomerpredominates. Even though one tautomer may be described, the presentinvention includes all tautomers of the present compounds.

The present invention also includes atropisomers of the presentinvention. Atropisomers refer to compounds of the invention that can beseparated into rotationally restricted isomers.

A compound of the invention, as described above, can be used in themanufacture of a medicament for the prophylactic or therapeutictreatment of a TGF-related disease state in an animal or human.

A compound of the invention is a potent inhibitor of transforming growthfactor (“TGF”)-β signaling pathway and are therefore of use in therapy.Accordingly, the present invention provides a method of preventing ortreating a TGF-related disease in an animal or human comprising the stepof administering a therapeutically effective amount of at least onecompound of the invention to the animal or human suffering from theTGF-related disease state.

As used herein, the term “therapeutically effective amount” refers to anamount of a compound of the invention required to inhibit the TGF-βsignaling pathway. As would be understood by one of skill in the art, a“therapeutically effective amount” will vary from patient to patient andwill be determined on a case by case basis. Factors to consider include,but are not limited to, the patient being treated, weight, health,compound administered, etc.

There are numerous disease states that can be treated by inhibition ofthe TGF-62 signaling pathway. Such disease states include, but are notlimited to, all types of cancer (e.g., breast, lung, colon, prostate,ovarian, pancreatic, melanoma, all hematological malignancies, etc.) aswell as all types of fibrotic diseases (e.g., glomerulonephritis,diabetic nephropathy, hepatic fibrosis, pulmonary fibrosis, arterialhyperplasia and restenosis, scleroderma, and dermal scarring).

The present invention also provides a pharmaceutical compositioncomprising at least one compound of the invention and at least onepharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier may be any such carrier known in the art including thosedescribed in, for example, Remington's Pharmaceutical Sciences, MackPublishing Co., (A. R. Gennaro edit. 1985). A pharmaceutical compositionof the invention may be prepared by conventional means known in the artincluding, for example, mixing at least one compound of the inventionwith a pharmaceutically acceptable carrier.

A pharmaceutical composition of the invention may be used in theprevention or treatment of a TGF-related disease state, as describedabove, in an animal or human. Thus, a compound of the invention may beformulated as a pharmaceutical composition for oral, buccal, intranasal,parenteral (e.g., intravenous, intramuscular or subcutaneous), topical,or rectal administration or in a form suitable for administration byinhalation or insufflation.

For oral administration, the pharmaceutical composition may take theform of, for example, a tablet or capsule prepared by conventional meanswith a pharmaceutically acceptable excipient such as a binding agent(e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); filler (e.g., lactose, microcrystallinecellulose or calcium phosphate); lubricant (e.g., magnesium stearate,talc or silica); disintegrant (e.g., potato starch or sodium starchglycolate); or wetting agent (e.g., sodium lauryl sulphate). The tabletsmay be coated by methods well known in the art. Liquid preparations fororal administration may take the form of a, for example, solution, syrupor suspension, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means with apharmaceutically acceptable additive such as a suspending agent (e.g.,sorbitol syrup, methyl cellulose or hydrogenated edible fats);emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicle (e.g.,almond oil, oily esters or ethyl alcohol); and preservative (e.g.,methyl or propyl p-hydroxybenzoates or sorbic acid).

For buccal administration, the composition may take the form of tabletsor lozenges formulated in conventional manner.

A compound of the present invention may also be formulated for sustaineddelivery according to methods well known to those of ordinary skill inthe art. Examples of such formulations can be found in U.S. Pat. Nos.3,538,214, 4,060,598, 4,173,626, 3,119,742, and 3,492,397, which areherein incorporated by reference in their entirety.

A compound of the invention may be formulated for parenteraladministration by injection, including using conventionalcatheterization techniques or infusion. Formulations for injection maybe presented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain a formulating agent such as a suspending,stabilizing and/or dispersing agent. Alternatively, the activeingredient may be in powder form for reconstitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

A compound of the invention may also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

For intranasal administration or administration by inhalation, acompound of the invention may be conveniently delivered in the form of asolution or suspension from a pump spray container that is squeezed orpumped by the patient or as an aerosol spray presentation from apressurized container or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. The pressurized containeror nebulizer may contain a solution or suspension of the compound of theinvention. Capsules and cartridges (made, for example, from gelatin) foruse in an inhaler or insufflator may be formulated containing a powdermix of a compound of the invention and a suitable powder base such aslactose or starch.

A proposed dose of a compound of the invention for oral, parenteral orbuccal administration to the average adult human for the treatment of aTGF-related disease state is about 0.1 mg to about 2000 mg, preferably,about 0.1 mg to about 200 mg of the active ingredient per unit dosewhich could be administered, for example, 1 to 4 times per day.

Aerosol formulations for treatment of the conditions referred to abovein the average adult human are preferably arranged so that each metereddose or “puff” of aerosol contains about 20 μg to about 10,000 μg,preferably, about 20 μg to about 1000 μg of a compound of the invention.The overall daily dose with an aerosol will be within the range fromabout 100 μg to about 100 mg, preferably, about 100 μg to about 10 mg.Administration may be several times daily, for example 2, 3, 4 or 8times, giving for example, 1, 2 or 3 doses each time.

Aerosol combination formulations for treatment of the conditionsreferred to above in the average adult human are preferably arranged sothat each metered dose or “puff” of aerosol contains from about 0.01 mgto about 1000 mg, preferably, about 0.01 mg to about 100 mg of acompound of this invention, more preferably from about 1 mg to about 10mg of such compound. Administration may be several times daily, forexample 2, 3, 4 or 8 times, giving for example, 1, 2 or 3 doses eachtime.

Aerosol formulations for treatment of the conditions referred to abovein the average adult human are preferably arranged so that each metereddose or “puff” of aerosol contains from about 0.01 mg to about 20,000mg, preferably, about 0.01 mg to about 2000 mg of a compound of theinvention, more preferably from about 1 mg to about 200 mg.Administration may be several times daily, for example 2, 3, 4 or 8times, giving for example, 1, 2 or 3 doses each time.

For topical administration, a compound of the invention may beformulated as an ointment or cream.

This invention also encompasses pharmaceutical compositions containingand methods of treatment or prevention comprising administering prodrugsof at least one compound of the invention. As used herein, the term“prodrug” refers to a pharmacologically inactive derivative of a parentdrug molecule that requires biotransformation, either spontaneous orenzymatic, within the organism to release the active drug. Prodrugs arevariations or derivatives of the compounds of this invention which havegroups cleavable under metabolic conditions. Prodrugs become thecompounds of the invention which are pharmaceutically active in vivo,when they undergo solvolysis under physiological conditions or undergoenzymatic degradation. Prodrug compounds of this invention may be calledsingle, double, triple etc., depending on the number ofbiotransformation steps required to release the active drug within theorganism, and indicating the number of functionalities present in aprecursor-type form. Prodrug forms often offer advantages of solubility,tissue compatibility, or delayed release in the mammalian organism (see,Bundgard, Design of Prodrugs, pp. 7–9, 21–24, Elsevier, Amsterdam 1985and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp.352–401, Academic Press, San Diego, Calif., 1992). Prodrugs commonlyknown in the art include acid derivatives well known to practitioners ofthe art, such as, for example, esters prepared by reaction of the parentacids with a suitable alcohol, or amides prepared by reaction of theparent acid compound with an amine, or basic groups reacted to form anacylated base derivative. Moreover, the prodrug derivatives of thisinvention may be combined with other features herein taught to enhancebioavailability. For example, a compound of the invention having freeamino, amido, hydroxy or carboxylic groups can be converted intoprodrugs. Prodrugs include compounds wherein an amino acid residue, or apolypeptide chain of two or more (e.g., two, three or four) amino acidresidues which are covalently joined through peptide bonds to freeamino, hydroxy or carboxylic acid groups of compounds of the invention.The amino acid residues include the 20 naturally occurring amino acidscommonly designated by three letter symbols and also include,4-hydroxyproline, hydroxylysine, demosine, isodemosine,3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid,citrulline homocysteine, homoserine, omithine and methionine sulfone.Prodrugs also include compounds wherein carbonates, carbamates, amidesand alkyl esters which are covalently bonded to the above substituentsof a compound of the invention through the carbonyl carbon prodrugsidechain.

According to the invention, in the treatment of a TGF-related diseasestate, a compound of the invention, as described herein, whether aloneor as part of a pharmaceutical composition may be combined with anothercompound(s) of the invention and/or with another therapeutic agent(s).Examples of suitable therapeutic agent(s) include, but are not limitedto, standard non-steroidal anti-inflammatory agents (hereinafterNSAID's) (e.g, piroxicam, diclofenac), propionic acids (e.g., naproxen,flubiprofen, fenoprofen, ketoprofen and ibuprofen), fenamates (e.g.,mefenamic acid, indomethacin, sulindac, apazone), pyrazolones (e.g.,phenylbutazone), salicylates (e.g., aspirin), COX-2 inhibitors (e.g.,celecoxib, valdecoxib, rofecoxib and etoricoxib), analgesics andintraarticular therapies (e.g., corticosteroids) and hyaluronic acids(e.g., hyalgan and synvisc), anticancer agents (e.g., endostatin andangiostatin), cytotoxic drugs (e.g., adriamycin, daunomycin,cis-platinum, etoposide, taxol, taxotere), alkaloids (e.g.,vincristine), and antimetabolites (e.g., methotrexate), cardiovascularagents (e.g., calcium channel blockers), lipid lowering agents (e.g.,statins), fibrates, beta-blockers, Ace inhibitors, Angiotensin-2receptor antagonists and platelet aggregation inhibitors, CNS agents(e.g., as antidepressants (such as sertraline)), anti-Parkinsonian drugs(e.g., deprenyl, L-dopa, Requip, Mirapex), MAOB inhibitors (e.g.,selegine and rasagiline), comP inhibitors (e.g., Tasmar), A-2inhibitors, dopamine reuptake inhibitors, NMDA antagonists, Nicotineagonists, Dopamine agonists and inhibitors of neuronal nitric oxidesynthase), anti-Alzheimer's drugs (e.g., donepezil, tacrine, COX-2inhibitors, propentofylline or metryfonate), osteoporosis agents (e.g.,roloxifene, droloxifene, lasofoxifene or fosomax), and immunosuppressantagents (e.g., FK-506 and rapamycin).

Biological Activity

The activity of the compounds of the invention for the variousTGF-related disease states as described herein can be determinedaccording to one or more of the following assays. According to theinvention, a compound of the invention exhibits an in vitro IC₅₀ valueof less than about 10 μM. For example, the compound of Example 5exhibits a TβRIIC₅₀ value of about 118 nM.

The compounds of the present invention also possess differentialactivity (i.e. are selective for) for TβRI over TβRII and TβRIII.Selectivity is measured in standard assays as a IC₅₀ ratio of inhibitionin each assay.

TGF-β Type II Receptor (TβRII) Kinase Assay Protocol

Phosphorylation of myelin basic protein (MBP) by the TβRII kinase wasmeasured as follows: 80 microliters of MBP (Upstate Biotechnology#13-104) diluted in kinase reaction buffer (KRB) containing 50 mM MOPS,5 mM MgCl₂, pH 7.2 to yield a final concentration of 3 micromolar MBPwas added to each well of a Millipore 96-well multiscreen-DP 0.65 micronfiltration plate (#MADPNOB50). 20 microliters of inhibitor diluted inKRB was added to appropriate wells to yield the desired finalconcentration (10–0.03 micromolar). 10 microliters of a mixture of ATP(Sigma #A-5394) and ³³P-ATP (Perkin Elmer #NEG/602H) diluted in KRB wasadded to yield a final concentration of 0.25 micromolar ATP and 0.02microcuries of ³³P-ATP per well. 10 microliters of a GST-TβRII fusionprotein (glutathione S-transferase at the N-terminal end of thecytoplasmic domain of TβRII-amino acids 193–567 with A to V change at438) diluted in KRB was added to each well to yield a finalconcentration of 27 nanomolar GST-TβRII. Plates were mixed and incubatedfor 90 minutes at room temperature. After the reaction incubation, 100microliters of cold 20% trichloroacetic acid (Aldrich #25,139-9) wasadded per well and plates were mixed and incubated for 60 minutes at 4°C. Liquid was then removed from the wells using a Millipore vacuummanifold. Plates were washed once with 200 microliters per well of cold10% trichloroacetic acid followed by two washes with 100 microliters perwell of cold 10% trichloroacetic acid. Plates were allowed to dryovernight at room temperature. 20 microliters of Wallac OptiPhaseSuperMix scintillation cocktail was added to each well. Plates weresealed and counted using a Wallac 1450 Microbeta liquid scintillationcounter. The potency of inhibitors was determined by their ability toreduce TβRII-mediated phosphorylation of the MBP substrate.

ALK-5 (TβRI) Kinase Assay Protocol

The kinase assays were performed with 65 nM GST-ALK5 and 84 nM GST-Smad3in 50 mM HEPES, 5 mM MgCl₂, 1 mM CaCl₂, 1 mM dithiothreitol, and 3_MATP. Reactions were incubated with 0.5_Ci of [33 P]_ATP for 3 h at 30°C. Phosphorylated protein was captured on P-81 paper (Whatman,Maidstone, England), washed with 0.5% phosphoric acid, and counted byliquid scintillation. Alternatively, Smad3 or Smad1 protein was alsocoated onto FlashPlate Sterile Basic Microplates (PerkinElmer LifeSciences, Boston, Mass.). Kinase assays were then performed inFlash-Plates with same assay conditions using either the kinase domainof ALK5 with Smad3 as substrate or the kinase domain of ALK6 (BMPreceptor) with Smad1 as substrate. Plates were washed three times withphosphate buffer and counted by TopCount (Packard Bio-science, Meriden,Conn.). (Laping, N.J. et al. Molecular Pharmacology 62:58–64 (2002)).

The following Examples illustrate the preparation of the compounds ofthe present invention. Melting points are uncorrected. NMR data arereported in parts per million (d) and are referenced to the deuteriumlock signal from the sample solvent (deuteriochloroform unless otherwisespecified). Mass Spectral data were obtained using a Micromass ZMD APCIMass Spectrometer equipped with a Gilson gradient high performanceliquid chromatograph. The following solvents and gradients were used forthe analysis. Solvent A; 98% water/2% acetonirile/0.01% formic acid andsolvent B; acetonitrile containing 0.005% formic acid. Typically, agradient was run over a period of about 4 minutes starting at 95%solvent A and ending with 100% solvent B. The mass spectrum of the majoreluting component was then obtained in positive or negative ion modescanning a molecular weight range from 165 AMU to 1100 AMU. Specificrotations were measured at room temperature using the sodium D line (589nm). Commercial reagents were utilized without further purification. THFrefers to tetrahydrofuran. DMF refers to N,N-dimethylformamide.Chromatography refers to column chromatography performed using 32–63 mmsilica gel and executed under nitrogen pressure (flash chromatography)conditions. Room or ambient temperature refers to 20–25° C. Allnon-aqueous reactions were run under a nitrogen atmosphere forconvenience and to maximize yields. Concentration at reduced pressuremeans that a rotary evaporator was used.

One of ordinary skill in the art will appreciate that in some casesprotecting groups may be required during preparation. After the targetmolecule is made, the protecting group can be removed by methods wellknown to those of ordinary skill in the art, such as described in Greeneand Wuts, “Protective Groups in Organic Synthesis” (2^(nd) Ed, JohnWiley & Sons 1991).

Analytical high performance liquid chromatography on reverse phase withmass spectrometry detection (LSMS) was done using Polaris 2×20 mm C18column. Gradient elution was applied with increase of concentration ofacetonitrile in 0.01% aqueous formic acid from 5% to 100% during 3.75min period. Mass spectrometer Micromass ZMD was used for molecular ionidentification.

EXAMPLE 1 Preparation of4-[3-(6-methyl-pyridin-2-yl)-isoxazol-4-yl]-quinoline

Step A: Preparation of[(6-Methyl-pyridyl-2-yl)-phenylamino-methyl]-phosphonic acid diphenylester

A 2 L round-bottom flask was charged with6-methyl-pyridine-2-carbalydehyde (40 g, 330 mmol), aniline (30.1 mL,330 mmol), and 380 mL of isopropyl acetate. The reaction mixture washeated to 65° C. and diphenylphosphite (112 mL, 495 mmol) was addeddropwise over 60 minutes. The mixture was stirred an additional 60minutes at 65° C., then at room temperature overnight. Concentration invacuo yielded a syrup that was dissolved in 1 liter of ethyl acetate andwashed with saturated sodium bicarbonate (3×200 mL). The organic layerwas dried over sodium sulfate, filtered and concentrated onto silicagel. Product was purified by flash chromatography using 10–20%ethylacetate/hexane solvent gradient to afford 126.5 grams of purematerial. HPLC t_(R)=6.68 min, LC-MS=431 (M+1).

Step B: Preparation of1-(6-Methyl-pyridyl-2-yl)-2-quinolin-4-yl-ethanone

A 2 L round-bottom flask was charged with[(6-Methyl-pyridyl-2-yl)-phenylamino-methyl]-phosphonic acid diphenylester (24.5 g, 57 mmol), quinoline-4-carbaldehyde (11.65 g, 74.1 mmol),90 mL of tetrahydrofuran, and 180 mL of isopropylalcohol. The reactionmixture was warmed to 25° C. and potassium t-butoxide (1.0M, 74.1 mL,74.1 mmol) was added dropwise over 90 minutes. After stirring anadditional 30 minutes, hydrochloric acid (2M, 122 mL) was added and themixture stirred one hour. The solution was heated to 45° C. and the pHwas adjusted to 5 with 6M sodium hydroxide, stirred an additional hour,then concentrated in vacuo. The residue was dissolved in chloroform andconcentrated onto silica gel. Product was purified by flashchromatography using 0–10% ethylacetate/hexane solvent gradient toafford 10.97 grams of pure material. HPLC t_(R)=5.02 min, LC-MS=263(M+1).

Step C: Preparation of4-[3-(6-Methyl-pyridyl-2-yl)-1H-pyrazol-4-yl-quinoline

A 10 mL round-bottom flask was charged with1-(6-Methyl-pyridyl-2-yl)-2-quinolin-4-yl-ethanone (100 mg, 0.38 mmol),1.5 mL of ethanol, and Hydroxylamine (140 uL, 2.3 mmol, 50% in water).This reaction mixture was stirred at room temperature for 24 hours.

LC-MS confirms the desired intermediate had formed. Next the reactionmixture was concentrated to complete dryness in vacuo. This residue wasdissolved in N,N-dimethylformamide dimethyl acetal (1.6 mL, 12 mmol)which was then stirred at 80° C. for 48 hours. LC-MS showed desiredproduct. The reaction mixture was concentrated to complete drynessagain. Product was purified by Shimodzu prep HPLC using a 5–100%acetonitrile/water solvent gradient (0.1% formic acid in both solvents,scan at λ=242 nm) to afford 18 mg of desired product. HPLC t_(R)=4.74min, LC-MS=288 (M+1).

EXAMPLE 26-[3-(6-Methyl-pyridin-2-yl)-isoxazol-4-yl]-[1,2,4]triazolo[4,3-a]pyridine

The title compound was prepared according to procedures analogous tothose described in Example 1. HPLC=3.71 (min). LC-MS=278 (M+1)

EXAMPLE 3 6-[3-(6-Methyl-pyridin-2-yl)-isoxazol-4-yl]-quinoxaline

The title compound was prepared according to procedures analogous tothose described in Example 1. HPLC=4.24 (min). LC-MS=289 (M+1)

EXAMPLE 4 4-Chloro-6-[3-(6-methyl-pyridin-2-yl)-isoxazol-4-yl]-quinoline

The title compound was prepared according to procedures analogous tothose described in Example 1. HPLC=4.87 (min). LC-MS=322 (M+1)

EXAMPLE 56-Methoxy-4-[3-(6-methyl-pyridin-2-yl)-isoxazol-4-yl]-quinoline

The title compound was prepared according to procedures analogous tothose described in Example 1. HPLC=4.64 (min). LC-MS=318 (M+1)

All publications, including but not limited to, issued patents, patentapplications, and journal articles, cited in this application are eachherein incorporated by reference in their entirety.

Although the invention has been described above with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A compound of formula (A):

or a pharmaceutically acceptable salt, tautomer, or hydrate thereof,wherein: X is O; R¹ is a group of the formula

 wherein R¹ can optionally be further independently substituted with atleast one moiety independently selected from the group consisting of:carbonyl, halo, halo(C₁–C₆)alkyl, perhalo(C₁–C₆)alkyl,perhalo(C₁–C₆)alkoxy, (C₁–C₆)alkyl, (C₂–C₆)alkenyl, (C₂–C₆)alkynyl,hydroxy, oxo, mercapto, (C₁–C₆)alkylthio, (C₁–C₆)alkoxy, (C₅–C₁₀)aryl,(C₅–C₁₀)aryloxy, (C₅–C₁₀)ar(C₁–C₆)alkyl, (C₅–C₁₀)ar(C₁–C₆)alkoxy,HO—(C═O)—, ester, amido, ether, amino, amino(C₁–C₆)alkyl,(C₁–C₆)alkylamino(C₁–C₆)alkyl, di(C₁–C₆)alkylamino(C₁–C₆)alkyl,(C₁–C₆)alkyl- and di(C₁–C₆)alkylamino, cyano, nitro, carbamoyl,(C₁–C₆)alkylcarbonyl, (C₁–C₆)alkoxycarbonyl, (C₁–C₆)alkylaminocarbonyl,di(C₁–C₆)alkylaminocarbonyl, (C₅–C₁₀)arylcarbonyl,(C₅–C₁₀)aryloxycarbonyl, (C₁–C₆)alkylsulfonyl, and (C₅–C₁₀)arylsulfonyl;each R³ is independently selected from the group consisting of:hydrogen, halo, halo(C₁–C₆)alkyl, (C₁–C₆)alkyl, C₂–C₆)alkenyl,(C₂–C₆)alkynyl, perhalo(C₁–C₆)alkyl, phenyl, (C₃–C₁₀)cycloalkyl,hydroxy, (C₁–C₆)alkoxy, perhalo(C₁–C₆)alkoxy, phenoxy,(C₃–C₁₀)cycloalkyl-O—, (C₁–C₆)alkyl-S—, (C₁–C₆)alkyl-SO₂—,(C₁–C₆)alkyl-NH—SO₂—, O₂N—, NC—, amino, Ph(CH₂)₁₋₆HN—, (C₁–C₆)alkyl HN—,(C₁–C₆)alkylamino, [(C₁–C₆)alkyl]₂-amino, (C₁–C₆)alkyl-SO₂—NH—,amino(C═O)—, aminoO₂S—, (C₁–C₆)alkyl-(C═O)—NH—,(C₁–C₆)alkyl-(C═O)—[((C₁–C₆)alkyl)-N]—, phenyl-(C═O)—NH—,phenyl-(C═O)—[((C₁–C₆)alkyl)-N]—, (C₁–C₆)alkyl-(C═O)—, phenyl-(C═O)—,C₃–C₁₀)cycloalkyl-(C═O)—, HO—(C═O)—, (C₁–C₆)alkyl-O—(C═O)—, H₂N(C═O)—,(C₁–C₆)alkyl-NH—(C═O)—, [(C₁–C₆)alkyl]₂-N—(C═O)—, phenyl-NH—(C═O)—,phenyl-[((C₁–C₆)alkyl)-N]—(C═O)—, (C₃–C₁₀)cycloalkyl-NH—(C═O)— and(C₁–C₆)alkyl-(C═O)—O—; where alkyl, alkenyl, alkynyl, phenyl,cycloalkyl, alkoxy, phenoxy, and amino of R³ is optionally substitutedby at least one substituent independently selected from (C₁–C₆)alkyl,(C₁–C₆)alkoxy, halo(C₁–C₆)alkyl, halo, H₂N—, Ph(CH₂)₁₋₆HN—, and(C₁–C₆)alkylHN—; s is an integer from one to five; and R⁴ is selectedfrom the group consisting of: hydrogen, halo, halo(C₁–C₆)alkyl,(C₁–C₆)alkyl, (C₂–C₆)alkenyl, (C₂–C₆)alkynyl, perhalo(C₁–C₆)alkyl,phenyl, (C₃–C₁₀)cycloalkyl, hydroxy, (C₁–C₆)alkoxy,perhalo(C₁–C₆)alkoxy, phenoxy, (C₃–C₁₀)cycloalkyl-O—, (C₁–C₆)alkyl-S—,(C₁–C₆)alkyl-SO₂—, (C₁–C₆)alkyl-NH—SO₂—, O₂N—, NC—, amino,Ph(CH₂)₁₋₆NH—, alkylNH—, (C₁–C₆)alkylamino, [(C₁–C₆)alkyl]₂-amino,(C₁–C₆)alkyl-SO₂—NH—, amino(C═O)—, aminoSO₂—,(C₁–C₆)alkyl-(C═O)—NH—,(C₁–C₆)alkyl-(C═O)—((C₁–C₆)alkyl)-N]—,phenyl-(C═O)—NH—, phenyl-(C═O)—((C₁–C₆)alkyl)-N]—, (C₁–C₆)alkyl-(C═O)—,phenyl-(C═O)—, cycloalkyl-(C═O)—, HO—(C═O)—, (C₁–C₆)alkyl-O—(C═O)—,H₂N(C═O)—, (C₁–C₆)alkyl-NH—(C═O)—, ((C₁–C₆)alkyl)₂-N—(C═O)—,phenyl-NH—(C═O)—,phenyl-((C₁–C₆)alkyl)-N]—(C═O)—,(C₃–C₁₀)cycloalkyl-NH—(C═O)— and(C₁–C₆)alkyl-(C═O)—O—; where alkyl, alkenyl, alkynyl, phenyl,cycloalkyl, alkoxy, phenoxy, and amino of R⁴ is optionally substitutedby at least one substituent independently selected from the groupconsisting of (C₁–C₆)alkyl, (C₁–C₆)alkoxy, halo(C₁–C₆)alkyl, halo, H₂N—,Ph(CH₂)₁₋₆—NH—, and (C₁–C₆)alkylNH—.
 2. A compound of claim 1, wherein sis one to two; R³ is hydrogen or (C₁–C₆)alkyl; and R⁴ is H,(C₁–C₆)alkyl, or (C₃–C₁₀)cycloalkyl.
 3. A pharmaceutical compositioncomprising a compound of claim 1 and a pharmaceutically acceptablecarrier.
 4. A compound;6-[3-(6-Methyl-pyridin-2-yl)-isoxazol-4-yl]-quinoxaline or apharmaceutically acceptable salt thereof.
 5. A pharmaceuticalcomposition comprising6-[3-(6-Methyl-pyridin-2-yl)-isoxazol-4-yl]-quinoxaline or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier.